Background:Staphylococcus aureus is Gram-positive bacterium commonly associated with nosocomial infections. The development of biofilm exhibiting drug resistance especially in foreign body associated infections has enabled the bacterium to draw considerable attention. However, till date, consensus guidelines for in vitro biofilm quantitation and categorization criterion for the bacterial isolates based on biofilm-forming capacity are lacking. Therefore, it was intended to standardize in vitro biofilm formation by clinical isolates of S. aureus and then to classify them on the basis of their biofilm-forming capacity. Materials and Methods: A study was conducted for biofilm quantitation by tissue culture plate (TCP) assay employing 61 strains of S. aureus isolated from clinical samples during May 2015– December 2015 wherein several factors influencing the biofilm formation were optimized. Therefore, it was intended to propose a biofilm classification criteria based on the standard deviation multiples of the control differentiating them into non, low, medium, and high biofilm formers. Results: Brain-heart infusion broth was found to be more effective in biofilm formation compared to trypticase soy broth. Heat fixation was more effective than chemical fixation. Although, individually, glucose, sucrose, and sodium chloride (NaCl) had no significant effect on biofilm formation, a statistically significant increase in absorbance was observed after using the supplement mix consisting of 222.2 mM glucose, 116.9 mM sucrose, and 1000 mM NaCl (P = 0.037). Conclusions: The present study puts forth a standardized in vitro TCP assay for biofilm biomass quantitation and categorization criteria for clinical isolates of S. aureus based on their biofilm-forming capacity. The proposed in vitro technique may be further evaluated for its usefulness in the management of persistent infections caused by the bacterium.

Staphylococcus aureus is a leading cause of morbidity and mortality in nosocomial and community-based infections.[1] It is associated with a number of infections ranging from dental caries, periodontitis, stye, carbuncle, impetigo, and pyoderma to persistent tissue infections such as wound infection, otitis media, osteomyelitis, rhinosinusitis, recurrent urinary tract infection, and endocarditis.[2] It is also one of the most important pathogens in implant-related infections.[3],[4] Several features of this bacterium render survival fitness in a wide variety of environments of which the biofilm formation is one of the special modes of persistent infections.[5],[6],[7],[8],[9],[10]

Biofilm formation is an adaptive protected mode of growth enabling bacteria to survive in hostile environments as in the human host. This mode also enables them to disperse and colonize new niches as per their need which is mediated by their chemical cross-talk called quorum sensing.[11],[12] The essential paradox of chronic infections is untreatability, and in most cases, chronic infections are accompanied by the formation of biofilms. The National Institute of Health, USA, claims the involvement of biofilms in 80% of all bacterial infections.[1] Neutrophil entrapment within biofilms leads to tissue injury by release of various inflammatory mediators. It has been observed that dead debris of neutrophils and/or other immune cells also serve as a biological matrix to facilitate biofilm formation. Bacterial genomic DNA liberated from biofilms is also an immunostimulant and is recognized by toll-like receptor 9.[12] Therefore, biofilms can be considered as a special mode of persistent bacterial infection.[13]

Further, biofilm formation is dependent on different parameters including the characteristics of the nature of carbon source, its concentration, pH, ionic strength, and temperature, etc.[14] Although investigators have tried to optimize the conditions required for biofilm formation by staphylococcal isolates, some of the parameters such as optimum concentration of sugars, salt, and richness of medium have not been thoroughly investigated.[15] Some investigators have used trypticase soy broth (TSB) with glucose and/or brain-heart infusion (BHI) broth with sucrose supplementations to assess the effect on biofilm phenotype.[16] However, some have comprehensively elucidated sodium chloride (NaCl) dependence of biofilms in S. aureus.[17] However, their quantitative interpretation and categorization based on biofilm production criteria were not clear and cannot be replicated in every laboratory settings. Therefore, a simple and consensus guideline for in vitro biofilm synthesis by clinical isolates of S. aureus is direly needed. To the best of our knowledge, the effect of growth medium, fixation and elution and then supplementation of different sugars and salt levels to a larger range of concentrations on the characteristics of S. aureus biofilm has received comparatively little attention as the majority of investigators have not screened the sugar and salt concentration beyond 1%.[14],[18] Further, there is no method described till date by which the bacteria can be differentiated on the basis of their biofilm-forming ability.

Therefore, in the present study, we aimed for the standardization of consensus protocol for achieving maximum in vitro biofilm formation by clinical isolates of S. aureus utilizing the supplementation with the proper concentration of glucose, sucrose, and NaCl. We also tried to put forth categorization criteria for the bacterial isolates on the basis of their biofilm-forming capacity.

Materials and Methods

Bacterial isolates

A study was conducted in which a total of 61 non-repetitive, consecutive strains of S. aureus isolated from the clinical samples received in the Microbiology laboratory over a period of 7 months (May 2015– December 2015), from various outpatients (outpatient departments [OPDs]) and inpatients wards of University Hospital, Banaras Hindu University. Of all the clinical isolates, majority were isolated from samples received from the Dermatology and Venereology OPD (n = 17), surgery OPD (n = 17), orthopedics ward (n = 10), high dependency unit (n = 4), pediatrics ward (n = 3), Intensive Care Unit (ICU) (n = 2), Neonatal ICU (n = 2), and one each from obstetrics and gynecology, plastic surgery, otorhinolaryngology, neurology, medicine, and urology wards [[Figure 1] and Supplementary Data 1] [Additional file 1].

Figure 1: Distribution pattern of isolates of Staphylococcus aureus from different outpatient departments and wards

Standardization of in vitro synthesis of biofilm in tissue culture grade microtiter plates

In the present study, the effect of various parameters on in vitro biofilm synthesis was at first observed on S. epidermidis American Type Culture Collection (ATCC) strains and S. aureus clinical isolates using 96-well flat bottom TCP.

Initial inoculum, media, and incubation

In the first step, we evaluated the effect of growth conditions for the preparation of initial inoculum (solid medium BHI agar vs. liquid medium TSB), effect of nutritional media for generation of biofilm (TSB vs. BHI broth), and incubation time (6, 12, 18, and 24 h) at 37°C.

In the first method, briefly fresh isolates were inoculated in TSB and BHI broth in stationary condition overnight at 37°C and diluted 1 in 100 with fresh medium for subsequent use. Each well of TCP was filled with 200 μl aliquots of the diluted cultures and then investigated for biofilm formation after 6, 12, 18, and 24 h at 37°C.

While in another method, the isolates were grown on BHI agar overnight at 37°C. Then, colonies from overnight grown BHI agar culture plates were suspended directly into physiological saline (0.89% NaCl), and vortexed to achieve a suspension of 0.5-McFarland turbidity (1.5 × 108 CFU/ml). Each well of TCP was filled with 190 μl aliquots of BHI and then 10 μl of bacterial suspension was added to it. Like above, the plates were read after 6, 12, 18, and 24 h of incubation.

Fixation

After respective incubations, the plates were inverted and gently tapped to remove residual broth. The wells were washed thrice with 200 μl of phosphate buffer saline (PBS) (pH 7.2) to remove planktonic bacteria before fixation.

The two protocols as mentioned above were compared for fixation of cells in the plates by two different methods. In the first method, cells were fixed with 200 μl of sodium acetate (2% w/v) for 30 min, while in another, plates were incubated for heat fixation at 60°C for 20 min. After fixation, the plate with sodium acetate was washed with 200 μl PBS thrice before staining.

Staining and elution

For staining, we used 175 μL of 0.5% crystal violet for 5 min. The excess crystal violet was removed, and the plates were washed with running tap water until runoff was clear. For elution, we used 150 μl ethanol-acetone mixture (80:20) and left at room temperature for 30 min. The elute was then resuspended in wells of new TCP to take optical density (OD) readings at λmax 550 nm in ELISA plate reader (Thermo Scientific, USA).

Supplementation with sugars and salt

Glucose, sucrose, and NaCl in different molar concentrations, namely, 55.6, 111.11, 166.7, and 222.2 mM for glucose; 29.2, 58.5, 116.9, and 175.4 mM for sucrose; and 500, 750, and 1000 mM for NaCl, respectively, were investigated to observe for any possible effect on the biofilm formation individually.

Based on the observations of maximum biofilm yielded by supplementation of the individual ingredient, a solution of optimum concentrations of glucose, sucrose, and NaCl (supplement mix) was selected to supplement the above method and the optimized method was then applied on all the clinical isolates once again.

Categorization of isolates based on biofilm-forming capacity

The following criteria were used for biofilm gradation in clinical isolates.

In this study, sterile broth and S. epidermidis ATCC 12228 served as the negative control. However, S. epidermidis ATCC 35984 (high slime producer) and ATCC 35983 (moderate slime producer) were used as positive control. All experiments with clinical isolates were done in quadruplet, i.e., each isolate were inoculated in four wells simultaneously and repeated thrice (on different days), and then, OD values were averaged and SD was calculated.

Statistical analysis

One-way ANOVA and one-tail t-test assuming equal variance were used to compute and analyze the differences in OD values obtained with different experimental variables of the in vitro synthesis of biofilm by TCP method. MS Excel data analysis tool along with IBM SPSS version 21.0, Armonk, New York was utilized for analysis. P≤ 0.05 was considered statistically significant.

Results

The following results were observed for different variables on in vitro biofilm synthesis by TCP assay in achieving conditions required for maximum biofilm biomass.

Effect of growth medium for harvesting bacterium for inoculum preparation

Higher biofilm formation was observed as inferred from increased OD when initial bacterial inoculum was prepared from the growth on BHI agar as compared to those grown in broths [Table 1].

The absorbance was significantly higher when BHI broth was used as the nutritional medium as compared to TSB (P = 0.00019, P< 0.05) [[Figure 2] and Supplementary Data 2] [Additional file 2]. For instance, the average OD for S. epidermidis ATCC 35984 was 1.491 ± 0.017 (OD ± SD) in BHI broth, which was 34% higher when compared with average OD in TSB (0.986 ± 0.019). Therefore, BHI broth was selected as the medium for characterization of biofilm formation of clinical isolates of S. aureus in the present study.

When ATCC control strains were assessed for the effect of incubation period on biofilm formation, maximum biofilm yield was found after 24 h with resultant average OD 0.991 ± 0.021 for ATCC 35984, 0.433 ± 0.012 for ATCC 35983, and 0.102 ± 0.017 for ATCC 12228. It was observed that after 6 h of incubation, the majority of the S. aureus isolates displayed insignificant absorbances with average OD ranging from 0.147 ± 0.0301 to 0.236 ± 0.0410. After 18 h, all isolates were found to produce biofilms as reflected by relative absorbances. The average OD for one of the isolates of S. aureus (Isolate number 27) was 0.358 ± 0.04, 0.511 ± 0.02, and 0.726 ± 0.04 at 12, 18, and 24 h, respectively. The similar pattern was also observed for other isolates. Statistically significant (P = 0.0015) results were observed after 24 h of incubation compared to 18 h of incubation and therefore was considered as the optimum incubation period for the assessment of biofilm-forming capacity of S. aureus [[Figure 3] and Supplementary Data 3] [Additional file 3].

Figure 3: Effect of incubation period on absorbance by clinical isolates of Staphylococcus aureus

When ATCC control strains were assessed for fixation by heat, it was found that there is a statistically significant increase in the absorbance as compared to sodium acetate fixation (P = 0.004) with average resultant OD 1.491 ± 0.017 for ATCC 35984, 0.478 ± 0.016 for ATCC 35983, and 0.129 ± 0.014 for ATCC 12228. However, with sodium acetate, average absorbance was found to be 0.973 ± 0.016 for ATCC 35984, 0.311 ± 0.021 for ATCC 35983, and 0.073 ± 0.017 for ATCC 12228.

It was observed that most of the clinical isolates displayed a perceivable biofilm-positive phenotype when BHI broth was supplemented with glucose [Supplementary Data 4] [Additional file 4]. Glucose in almost all concentrations was positively added to the biofilm formation, but highest absorbance was observed at 222.2 mM glucose. However, individual concentrations of glucose had no significant effect on absorbance (P = 0.135) [Figure 4].

Figure 4: Effect of different concentrations of glucose supplementations on absorbance

It was noted that less number of clinical isolates displayed a biofilm-positive phenotype when BHI broth was supplemented with sucrose (P = 0.21). Sucrose also had no significant effect on absorbance. However, it has shown maximum absorbance at concentration of 116.92 mM. Beyond 116.92 mM concentration saturation was observed and in some cases, even the loss in the biofilm was observed as reflected by ODs [[Figure 5] and Supplementary Data 4].

Figure 5: Effect of different concentrations of sucrose supplementations on absorbance

Based on the observations of different variables of in vitro biofilm synthesis including sugars and NaCl concentration as described above, all the stains were subjected to biofilm formation on the selected combination of 222.2 mM glucose, 116.9 mM sucrose, and 1000 mM NaCl (supplement mix). A significant increase in the biofilm formation (P = 0.031) was observed after supplementation as compared to unsupplemented BHI broth [[Figure 7], [Figure 8] and Supplementary Data 6] [Additional file 6].

Categorization of Staphylococcus aureus isolates based on biofilm-forming capacity

We tried to establish criteria for categorizing S. aureus isolates based on their biofilm-forming capacity. Based on the results obtained from TCP assay with supplement mix, a cut-off OD (ODcut) was obtained by taking the average of all the ODs of the negative control ATCC 12228 and thrice the value of SD of the negative control was added to it.

In this study, the average OD of the negative control came to be 0.147 ± 0.0305. Hence, the cutoff OD value in the current study was set as 0.238. The isolates which have OD value lesser than 0.238 were considered as NBFs [Table 2].

Table 2: Categorization of biofilm made by strains of Staphylococcus aureus (n=61)

Table 3: Distribution of isolates in different classes in toto and selective distribution of methicillin-susceptible Staphylococcus aureus and methicillin-resistant Staphylococcus aureus isolates in different classes

Effect of opting Staphylococcus epidermidis ATCC 12228 as the negative control

It was observed that the ODs of moderate and high biofilm producing ATCC strains of S. epidermidis lied repeatedly in the range of 2 × ODcut < OD ≤4 × ODcut and OD >4 × ODcut respectively with respect to the non-former ATCC 12228 strain. Therefore, opting S. epidermidis ATCC 12228 as the negative control was considered to be more useful in deciding the precise cut-off criteria rather than the broth alone [Figure 8] and [Table 1].

The optimized protocol for the in vitro synthesis of biofilm by TCP assay for clinical isolates of S. aureus has been summarized in [Figure 9].

Out of 61 clinical isolates of S. aureus, 18 (29.51%) were MRSA. The majority of S. aureus isolates were found to be resistant to more than 9 antibiotics. All the clinical isolates were found to be sensitive to linezolid and netilmicin. Only 3 isolates were penicillin sensitive. Isolates have shown lesser susceptibility toward ciprofloxacin as the majority was either resistant or intermediate susceptible. The majority of isolates (n = 37) showed intermediate resistance to the erythromycin. However, compared to azithromycin, the incidence of resistance was lesser with erythromycin. Most of the isolates (n = 44) were resistant to co-trimoxazole. Four isolates were resistant to fusidic acid while mupirocin resistance was detected in only one strain [Supplementary Data 1]. Strong and moderate biofilm-producing isolates were found to be more resistant to commonly used antibiotics compared to weak producing ones [Table 4].

Table 4: Biofilm-forming ability of strains of different resistance pattern

Biofilm is a sessile microbial community wherein cells are attached to a surface (biotic or abiotic) and are enmeshed within a self-produced protective extracellular polymeric matrix. This extracellular polymeric matrix in S. aureus/S. epidermidis is poly-N-acetyl glucosamine (PNAG).[20] There are cases where PNAG-independent proteinaceous biofilms are also reported in S. aureus.[21],[22]

Schleifer and Kroppenstedt reported the surface association of the infecting bacteria and speculated similarity of solid agar grown bacteria to natural infection settings and then to the pathogens grown in liquid media.[23] When initial inoculum was prepared from the bacteria grown on BHI agar, we noticed their comparatively higher efficiency in biofilm production as compared to those grown in broths. This could be probably a result of the higher expression of surface proteins required for adherence when bacteria are grown on solid media. The expression of these proteins is also reported as a prerequisite for infectivity in various studies.[24]

The richness of nutrients is another important factor which influences the ability of bacteria to produce biofilm.[15] Some investigators have utilized TSB for biofilm quantitation.[25],[26] In the current study, BHI broth was found to be significantly more effective in biofilm formation [Supplementary Data 2]. Proteins especially rich in leucine, proline, serine, and aspartate are abundant in BHI broth since these amino acids may be essential for the production of adhesins such as fibronectin-binding protein and clumping factors which are necessary for adherence. The presence of lipids such as choline and sphingosine in BHI may have added advantage in biofilm formation and provide resistance from desiccation. Further, it is a source of sugars such as inositol/myoinositol which cannot be fermented by S. aureus leading to resistance in pH fall, which, in turn, may be needed for robust biofilm architecture. These results indicate a strong dependence of biofilm formation in S. aureus and the environmental conditions required for growth, which seems to be even more pronounced in S. aureus than in S. epidermidis.[22],[27],[28],[29] Similarly, while observing the effect of incubation period on in vitro biofilm formation, it was noticed that after 6 h of incubation, the majority of the S. aureus isolates remained NBF and for some of the isolates biofilms were even non-detectable. Adhesion of bacterial cells to microtiter plate appeared to be a function of time and increased linearity was observed with time progression. Although biofilm formation was observed in all isolates after 18 h of incubation, the maximum biofilm yield as reflected in ODs was observed after 24 h of incubation as also noticed by other investigators.[15],[16]

The fixation of attached cells by heating at 60°C for 20 min was found to be statistically more significant than fixation by sodium acetate in our study. Therefore, we opted for heat fixation. Heat disrupts hydrogen bonds and non-polar hydrophobic interactions of bacterial cell surface proteins leading to coagulation and in some cases its denaturation. Further, it dehydrates the sugar content leading to the crude biomass estimation. While sodium acetate has a protective effect against denaturation.[30] These results are in consonance with the observations of Baldassarri et al.[31]

During elution step, only 150 μL of eluent (ethanol:acetone [80:20]) was added per well, to evade interference with the stained matter at the liquid– air interface, which is not considered to be indicative of biofilm formation.

We examined the biofilm formation in both MRSA and MSSA isolates in media supplemented with different concentration of glucose, sucrose, and NaCl. Although the addition of sugars and salts individually has increased the biofilm phenotype as manifested by an increase in OD, it was not statistically significant (P > 0.05). On the other hand, when the supplement mix was added to the broth in a defined ratio, the significant increase in OD was observed (P = 0.037, P < 0.05). Therefore, it is strongly recommended to use the proposed method for in vitro biofilm quantitation.

Among MSSA isolates, isolate-to-isolate variation was observed with respect to biofilm-forming ability with nature of supplementation used. Glucose in almost all concentrations was positively added to the biofilm formation while sucrose at concentration beyond 116.92 mM showed almost saturation and in some cases even the loss in the biofilm. NaCl at 1000 mM concentration showed the maximum increase in absorbance. This observation was found consistent with Lim et al. who found enhanced expression of rbf gene involved in the signal transduction pathway for biofilm production when the NaCl concentration is above 1.6% but not when it is below 1.6%.[17]

While observing biofilm synthesis by MRSA isolates, the strong correlation existed between the biofilm phenotype and the concentration of the sugar supplemented. Even some isolates showed exceptional behavior to this generalized rule [Supplementary Data 3],[Supplementary Data 4],[Supplementary Data 5]. Although this sort of heterogeneity in biofilm-forming capacity of MRSA has been addressed earlier, isolate-wise exceptional behavior has never been highlighted. Each isolate responded differently from one another regarding response to the sugar and, in turn, in biofilm phenotype. Pozzi et al. (2012) proposed that acquisition of methicillin resistance appears to repress polysaccharide-type biofilm production and promote the formation of proteinaceous biofilms as evidenced by biofilm phenotype observations made in the present study.[32],[33] However, there are certain MRSA isolates which showed the exception to this generalized rule. The universality to this generalized rule is just an enigma. Biofilm development in MRSA isolates is primarily glucose induced but not solely, and apparently, involves a protein adhesin.[21],[34]

Till date, there is no consensus view regarding categorization of S. aureus isolates based on their biofilm-forming capacity. Therefore, the definition of a strong, medium, weak, and non-biofilm producer varies greatly among the studies.[15],[16],[35],[36] Mathur et al. have recently proposed the criteria for grading the isolates based on their ability to form a biofilm which considered non-former isolates when the OD was <0.120, while OD range for medium-former was >0.120– ≤0.240 and for those of high former was >0.240.[16] Similarly, Stepanovic et al. have also proposed the criteria for biofilm classification and used the same old gold standard of Christensen et al. using the same ATCC 35984, 35983, and 12228 reference isolates.[15] Christensen et al. have used only an approximation of distance plotted in a graph, by dividing the graph into three portions: nonadherent (OD in both media, <0.120), weakly adherent (OD in either medium, >0.120 but 0.240), and strongly adherent (OD in either medium, >0.240).[37]

In the present study, a need of new cut-off criteria was felt because of the aforesaid reason and significant increase of the OD expanding the limit of OD in previously described non, moderate, and high biofilm-former category. A plethora of literature is available where only broth was taken as the negative control. In this study, S. epidermidis ATCC 12228 as the negative control was found to be more accurate in deciding the precise cutoff criteria rather than the broth alone. Broth can be used to ensure the sterility during the execution of the experiment. As negative and positive controls are a must in any experimental setup, we propose the OD cutoff criteria based on the OD of the negative control and the addition of some factor to its SD value. And then, various multiples (even) of ODcut can be used to distinguish clinical isolates based on their biofilm-forming capacities. By adopting the proposed method and criteria, it was observed that reference strains ATCC 35984, 35983, and 12228 remained in their respective classes as high, medium, and non-formers. However, it was interesting to observe that when the new criterion was applied on all the clinical isolates of S. aureus, all the previously declared nonformer isolates were either shifted to low former or to the medium-former category. Therefore, instead of using uninoculated broth, ATCC 12228 may be used as negative control for error free and concordant results. This method can, therefore, be unequivocally used for all clinical staphylococcal isolates to adapt the low/WBFs as reported by other investigators also.[15],[16],[28],[38]

It was observed that ODs of a number of clinical isolates of S. aureus lied between the non and the moderate biofilm range. Therefore, a new category of WBFs is needed to be introduced in the study of biofilm quantitation and also for the sake of uniformity. To further strengthen the validity of results on biofilm quantitation, one may need a higher number of reference strains of both S. epidermidis as well as S. aureus of all the four grades of biofilm producers.

In the present study, strong and MBF isolates were found to be more resistant to commonly used antibiotics compared to WBFs. Strong biofilm producers are more adapted pathogenic strains and have acquired resistance over the period due to continuous exposure to the antibiotics or by acquiring genes through horizontal gene transfer or by both. This may be the consequence of biofilm providing an appropriate environment for the transfer of drug resistance determinants.[39]

Further, investigators claimed that as much as thousand times increased MIC of biofilm-dwelling cells than the planktonic cells.[1] This may be due to interruptions posed by the biofilm slimy matrices in the form of electrostatic repulsion and/or sequestration of antibacterial substances apart from being diffusion barrier.[40],[41] There are attempts which were made to design a number of anti-biofilm compounds mainly short peptides, which seems to be promising strategy against staphylococcal biofilm.[42] However, in the future, for these and several other candidate drugs, there will be a need for a standardized method for in vitro biofilm synthesis by S. aureus along with classification criterion for conclusive authentication of drugs as potential antibiofilm agents.

However, the limitation of the current study is that the method of biofilm formation proposed here may not be useful for Gram-negative isolates. This is because, among Gram-negative bacteria, altogether, different operon arrays are responsible for controlling biofilm biogenesis. In Gram-negative bacteria, some of the polysaccharides are neutral or polyanionic due to the presence of uronic acids or ketal-linked pyruvates.[40] However, classification criteria can be used with properly established negative control.

Conclusions

The results indicate that the different variables including supplement mix containing glucose, sucrose, and NaCl in a defined ratio enhances the biofilm-forming ability of S. aureus significantly in the proposed method of in vitro biofilm formation assay employing TCP. The present study puts forth a standardized in vitro TCP assay for biofilm synthesis by S. aureus and its categorization indicating their differential ability to produce biofilm. The proposed in vitro technique may be further evaluated for its usefulness in the management of persistent infections caused by the bacteria.

Financial support and sponsorship

The present work is accomplished by the grant sanctioned as contingency grant offered to AKS as JRF by University Grants Commission, New Delhi, and DST-PURSE GRANT sanctioned to Department of Microbiology, Institute of Medical Sciences, Banaras Hindu University.